The polymorphonuclear leukocyte (PMN) is the primary cellular component of the acute inflammatory response. PMN migrate from the bloodstream to a site of inflammation where they generate toxic, reduced metabolites of O2 (O2-, H2o2, OH) and release destructive enzymes (peroxidase, lysosomal hydrolases) into phagocytic vacuoles or extracellularly. Elucidation of the mechanisms by which the production and release of these agents are regulated is of prime importance to th understanding of host-defense mechanisms and to allowing the potential pharmacological manipulation of these processes to the benefit of the host. The present research proposal is designed to test the hy0othesis that activation of PMN by diverse stimuli produces an elevated cytoplasmic free Ca2+ which then affect PMN functions by Ca2+ + calmodulin regulation of intracellular enzymes. This program focuses on activation of NAD(P)H oxidase and degranulation as two major processes which are stimulated in PMN separately or together by specific stimuli. Experiments are designed to determine the magnitude of cytoplasmic Ca2+ changes, the source of this Ca2+ and mechanisms of regulation of Ca2+ transients in activated PMN. Attempts to relate alteratons in cell Ca2+ levels to function will be made using Ca2+-depleted and restored cells and the critical use of agents which may either stimulate (ionophore A23187) or inhibit (verapamil, TMB-8) Ca2+ fluxes or inhibit Ca2+ calmodulin interactions with proteins (trifluoperazine, W-7). The effects of elevated intracellular Ca2+ on the phosphorylation and methylation states of membranous and soluble proteins and methylation of phosphoipids in membranes of whole cells will be examined. Studies with isolated subcellular fractions (plasma membranes, granule and soluble fractions) will determine whether Ca2+ + calmodulin may activate protein-kinases or -methylases or phospholipid methyltransferases. Alterations in these parameters will be related to NAD(P)H oxidase activity and granule-plasma membrane fusion and enzyme release both in intact cells and subcellular fractions. These studies will determine whether interaction of Ca2+ with calmodulin provides a mechanism common to activation of PMN by diverse stimuli. They will further define the role of two established means of cellular regulation: phosphorylationand methylation, in PMN activation and their potential regulation by Ca2+.